The overall hypothesis of this grant is that acute (less than 120 min) actions of estrogen on UA endothelial cells (UAEC) upon binding to membrane estrogen receptor (ER) lead to eNOS phosphorylation and dissociation from caveolin-1 as well as association with heat shock protein 90 (HSP90) thereby increasing eNOS activity directly and/or indirectly via the mitogen- activated protein kinase (MAPK) pathway. Whereas, chronic (greater than 2hr) estrogen actions on UAEC result in down- regulation of UA endothelial caveoln-1 expression, and this is ER-mediated and at least in part through membrane ER-mediated activation of MAPK which translocates into the nucleus to stimulate the AP-1 transcription factors thereby inhibiting caveolin-1 expression. Furthermore, there are direct relationships between changes in caveolin-1 HSP90 associated eNOS and caveolin-1 levels in UA endothelium and rises in UBF during the estrous cycle and ERT and pregnancy. To address this hypothesis, the following specific aims will be studied using sheep UA endothelium as the experimental target. Specific aim 1: to further characterize activation of extracellular signal- regulated kinases (ERK2/1) and other MAPK family members (JNK, p38mapk, and ERK5) by estrogen and to determine the membrane ER- initiated signaling that results in ERK activation in response to estrogen and the membrane impermeable E2b-BSA in UAEC. Specific aim 2: to establish the time- and dose-dependency of estrogen stimulated eNOS activity, NO production, eNOS phosphorylation and dissociation from caveolin-1 as well as association with HSP90 in UAEC. Specific aim 3: to establish if estrogen stimulation of NO production in UAEC is through MAPK phosphorylation of eNOS and eNOS dissociation from caveolin-1 and association with HSP90 via the MAPK pathway. Specific aim 4: to establish in UAEC if estrogen activated MAPK translocates into the nucleus to stimulate the AP-1 (Fos/Jun dimers) transcription factors thereby down-regulating caveolin-1 expression, and if down-regulation of caveolin-1 is associated with reduced or lost of caveolae. Specific aim 5: to establish if the amounts of UA endothelial caveolin-1/HSP90 associated eNOS changes in vivo during short- term (0-120 min) ERT, and UA endothelial caveolin-1 expression is altered in vivo during long-term (days) ERT, ovarian cycle, and pregnancy, and if these changes are associated with rises in UBF. UBF increases substantially during pregnancy in order to provide sufficient oxygen and nutrient supply for the development of the growing fetus. Insufficient blood supply during pregnancy can result in IUGR, preeclampsia, and decreased neonatal birthweight, which in turn inversely correlates to neonatal morbidity. Thus, this grant application will provide important clinical implications in perinatal healthcare. It may also have relevant implications in the systemic cardiovascular system since estrogen is believed to be the major reason that young women have lower risk in cardiovascular diseases than men.